2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine found in cooked meat, induces mammary gland cancer when administered to adolescent female rats (43-day-old). In contrast, mature virgin rats (150-day-old) were resistant to mammary carcinogenesis by PhIP. To explore the possible mechanisms for the age-related differences in susceptibility, PhIP-DNA adduct levels, mutations, and gene expression were examined in glands from 43-day and 150-day-old PhIP-treated rats. In rats of different ages, PhIP-DNA adduct levels detected by the postlabeling assay and mutant frequency measured in the lacI reporter gene of Big Blue rats were not statistically different. PhIP-DNA adduct levels, adduct removal, and mutation burden did not appear to account for the variation in carcinogen susceptibility with age. However, cDNA microarray analysis indicated that PhIP treatment differentially altered the profile of gene expression in glands from 43-day-old and 150-day-old rats. In 150-day-old rats, PhIP enhanced the expression of genes associated with differentiation (eg, beta-casein, kappa-casein, whey acidic protein) and induced morphological differentiation. In contrast, in 43-day-old rats, PhIP inhibited the expression of differentiation genes and enhanced cellular proliferation. From 3 hours to 6 weeks after PhIP dosing, the number of clones showing altered expression declined more than 50% in 150-day-old rats but increased fourfold in 43-day-old rats (29 clones versus 194, respectively) suggesting that PhIP induced a cascade of gene expression alterations only in susceptible rats. Genes showing altered expression specifically in 43-day-old rats included the Ras superfamily genes and genes associated with protein synthesis/degradation (lysosomal proteins, heat shock proteins, and proteasomes). The microarray data support the notion that the mechanism of age-dependent susceptibility to mammary gland cancer is largely associated with differential responses in expression of genes involved in cellular differentiation, proliferation, and protein homeostasis.Identification of molecular markers of early-stage breast cancer development is important for the diagnosis and prevention of the disease. In the present study, we used microarray analysis to examine the differential expression of genes in the rat mammary gland soon after treatment with a known chemical carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA), and prior to tumor development. Six weeks after DMBA, differential expression of multiple genes involved in cell growth, differentiation and microtubule dynamics were observed. Gene expression changes were further validated by a combination of techniques, including real-time PCR, RT-PCR, Western blotting and immunohistochemistry. An inhibition of differentiation in this early stage was suggested by the lower expression of beta-casein and transferrin and higher expression of hsp27 in glands from DMBA-treated rats. Possible cell cycle deregulation was indicated by an increased expression of cyclin D1 and hsp86, a heat shock protein associated with cyclin D1. Prior to tumor development, DMBA increased cellular proliferation as detected by Ki-67 and stathmin immunostaining in histologically normal mammary gland. Genes regulating microtubule function, including stathmin, Ran, alpha-tubulin and hsp27, were all overexpressed in the mammary gland of DMBA-treated rats, raising the possibility that disruption of microtubule dynamics and abnormal mitosis may be critical events preceding breast cancer development. Several of the altered proteins, including hsp27, hsp86 and stathmin, may ultimately serve as markers of early breast cancer development.